1.1 Field of the Invention
The present invention relates generally to the fields of molecular biology. More particularly, certain embodiments concern methods and compositions comprising DNA segments, and proteins derived from bacterial species. More particularly, it concerns a novel cryET29 gene from Bacillus thuringiensis encoding a coleopteran- and cat flea-toxic crystal protein. Various methods for making and using these DNA segments, DNA segments encoding synthetically-modified CryET29 proteins, and native and synthetic crystal proteins are disclosed, such as, for example, the use of DNA segments as diagnostic probes and templates for protein production, and the use of proteins, fusion protein carriers and peptides in various immunological and diagnostic applications. Also disclosed are methods of making and using nucleic acid segments in the development of transgenic plant cells containing the DNA segments disclosed herein.
1.2 Description of the Related Art
1.2.1 Bacillus thuringiensis Crystal Proteins
Bacillus thuringiensis is a Gram-positive bacterium that produces δ-endotoxins known as crystal proteins which are specifically toxic to certain orders and species of insects. Many different strains of B. thuringiensis have been shown to produce insecticidal crystal proteins. Compositions including B. thuringiensis strains which produce insecticidal proteins have been commercially available and used as environmentally acceptable insecticides because they are quite toxic to the specific target insect, but are harmless to plants and other non-targeted organisms.
The B. thuringiensis crystal protein is toxic in the insect only after ingestion when the alkaline pH and proteolytic enzymes in the insect mid-gut solubilize the crystal protein and release the toxic components. These components disrupt the mid-gut cells causing the insect to cease feeding and, eventually to die. In fact, B. thuringiensis has proven to be an effective and environmentally safe insecticide in dealing with various insect pests.
As noted by Hofte et al., (1989) the majority of insecticidal B. thuringiensis strains are active against insect of the order Lepidoptera, i.e., caterpillar insects. Other B. thuringiensis strains are insecticidally active against insects of the order Diptera, i.e., flies and mosquitoes, or against both lepidopteran and dipteran insects. In recent years, a few B. thuringiensis strains have been reported as producing crystal proteins that are toxic to insects of the order Coleoptera, i.e., beetles. To date, there have been no reports of B. thuringiensis strains active on fleas of the Genus, Ctenocephalides, in the order Siphonaptera.
The dipteran-active Cyt toxins differ from most of the other B. thuringiensis insecticidal crystal proteins in that they are smaller and do not share conserved blocks of sequence homology. These proteins demonstrate broad cytolytic activity in vitro, yet are specifically toxic to larvae of dipteran insects in vivo. These properties have been described elsewhere (Chilcott and Ellar, 1988).
1.2.2 Genetics of Crystal Proteins
A number of genes encoding crystal proteins have been cloned from several strains of B. thuringiensis. The review by Höfte et al. (1989) discusses the genes and proteins that were identified in B. thuringiensis prior to 1990, and sets forth the nomenclature and classification scheme which has traditionally been applied to B. thuringiensis genes and proteins. cryI genes encode lepidopteran-toxic CryI proteins. cryII genes encode CryII proteins that are toxic to both lepidopterans and dipterans. cryIII genes encode coleopteran-toxic CryIII proteins, while cryIV genes encode dipteran-toxic CryIV proteins.
Recently a new nomenclature has been proposed which systematically classifies the cry genes based upon DNA sequence homology rather than upon insect specificities. This classification scheme is shown in Table 1.
TABLE 1Revised B. thuringiensis δ-Endotoxin Gene NomenclatureaNewOldGenBank Accession #Cry1AaCryIA(a)M11250Cry1AbCryIA(b)M13898Cry1AcCryIA(c)M11068Cry1AdCryIA(d)M73250Cry1AeCryIA(e)M65252Cry1BaCryIBX06711Cry1BbET5L32020Cry1BcPEG5Z46442Cry1CaCryICX07518Cry1CbCryIC(b)M97880Cry1DaCryIDX54160Cry1DbPrtBZ22511Cry1EaCryIEX53985Cry1EbCryIE(b)M73253Cry1FaCryIFM63897Cry1FbPrtDZ22512Cry1GPrtAZ22510Cry1HPrtCZ22513Cry1HbU35780Cry1IaCryVX62821Cry1IbCryVU07642Cry1JaET4L32019Cry1JbET1U31527Cry1KU28801Cry2AaCryIIAM31738Cry2AbCryIIBM23724Cry2AcCryIICX57252Cry3ACryIIIAM22472Cry3BaCryIIIBX17123Cry3BbCryIIIB2M89794Cry3CCryIIIDX59797Cry4ACryIVAY00423Cry4BCryIVBX07423Cry5AaCryVA(a)L07025Cry5AbCryVA(b)L07026Cry5BU19725Cry6ACryVIAL07022Cry6BCryVIBL07024Cry7AaCryIIICM64478Cry7AbCryIIICbU04367Cry8ACryIIIEU04364Cry8BCryIIIGU04365Cry8CCryIIIFU04366Cry9ACryIGX58120Cry9BCryIXX75019Cry9CCryIHZ37527Cry10ACryIVCM12662Cry11ACryIVDM31737Cry11BJeg80X86902Cry12ACryVBL07027Cry13ACryVCL07023Cry14ACryVDU13955Cry15A34 kDaM76442Cry16Acbm71X94146Cyt1ACytAX03182Cyt2ACytBZ14147To Be AssignedCryET29, Present InventionTo Be AssignedaAdapted from: Crickmore, N. et al. Microbiol. and Mol. Bio. Rev. (1998) Vol. 62: 807-813.1.2.3 Identification of Crystal Proteins Toxic to Coleopteran Insects
The cloning and expression of a gene encoding a 26-kDa mosquitocidal toxin from the dipteran-active B. thuringiensis var. israelensis has been described (Ward et al., 1984), and the nucleotide sequence of this gene was reported (Ward and Ellar, 1986). The molecular mass of the toxin protein, CytA, calculated from the deduced amino acid sequence was determined to be 27,340 Da.
The nucleotide sequence of the gene for a 27-kDa mosquitocidal Cyt protein isolated from B. thuringiensis var. morrisoni strain PG14 has been disclosed (Earp and Ellar, 1987). The sequence of this toxin protein was found to differ by only one amino acid residue from the CytIA protein of B. thuringiensis var. israelensis. 
The identification of a 25-kDa protein that exhibits cytolytic activity in vitro when activated by proteolysis from the mosquitocidal B. thuringiensis var. kyushuensis was described earlier (Knowles et al., 1992), and the nucleotide sequence of the gene for this protein, CytB, was reported (Koni and Ellar, 1993). The predicted molecular mass of the CytB protein is 29,236 Da and the deduced amino acid sequence is quite distinct, although it does share significant sequence similarity with the CytA protein of B. thuringiensis var. israelensis. 
The cloning and characterization of the gene for a 30-kDa toxin protein with activity on coleopteran and dipteran insects has been described (Intl. Pat. Appl. Pub. No. WO 95/02693, 1995). This gene, isolated from B. thuringiensis PS201T6, encodes a protein of 29,906 Da which exhibits a 64% sequence identity with the CytA toxin of B. thuringiensis var. israelensis. 